Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.     

Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5-kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin-hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the phosphatidylcholine-hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster. As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli. The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20-30-fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2.     

Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase-like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 micrograms of enzyme preparation. Dialysis of the homogenates against Tris-HCl and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.     

Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.

Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.     

Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.     

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.     

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.     

Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.     

Cloning and expression of rat neutral sphingomyelinase: enzymological characterization and identification of essential histidine residues

Using cross-species sequence homology, we cloned a cDNA for rat neutral sphingomyelinase (nSMase) composed of 422 amino acids that shares 87.6 and 79.0% identity with the mouse and human forms respectively. The rat nSMase expressed in Escherichia coli catalyzed sphingomyelin hydrolysis at neutral pH in a Mg(2+)-dependent manner, and required Triton X-100, dithiothreitol, and KCl for its full activity. The cloned rat enzyme shares conserved sequences with nSMases from both eukaryotes and prokaryotes. Introduction of single mutations into either of the histidine residues at positions 136 and 272, putative active sites, entirely abolished the activity, supporting a common mechanism for the nSMase family independent of the species. However, mutation in histidine 151, conserved only in eukaryotes, also abolished the activity, suggesting eukaryote-specific control of nSMase linked to this histidine 151. This enzyme also catalyzed the hydrolysis of lyso-platelet activating factor to yield 1-alkylglycerol at a rate that is slightly lower than that with sphingomyelin.     

Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties.

A sphingomyelinase was purified 980-fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)2SO4 precipitation and chromatography on CM-Sephadex, DEAE-cellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholipases. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (Mr = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.     

Molecular identification of family 38 alpha-mannosidase of Bacillus sp. strain GL1, responsible for complete depolymerization of xanthan

When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, alpha-mannosidase exhibiting activity toward p-nitrophenyl-alpha-D-mannopyranoside (pNP-alpha-D-Man) was produced intracellularly. The 350-kDa alpha-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-alpha-D-Man (Km = 0.49 mM) and D-mannosyl-(alpha-1,3)-D-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for alpha-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of alpha-mannosidases belonging to glycoside hydrolase family 38.     

Sphingomyelinase of chicken erythrocyte membranes.

Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [³H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg²⁺ and Mn²⁺ activate the enzyme while Ca²⁺ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% ($\text{w}\text{v}$) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.     

A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium,Alteromonas sp. strain O-7

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.     

Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.     

Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.     

Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family

Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.